Methods for isolating mutant microorganisms from parental populations

ABSTRACT

A method for isolating a mutant microorganism is described. The method comprises the steps of: (a) separately microencapsulating in a semi-permeable membrane each or a small number of microorganisms from a microorganism population containing said mutant; (b) growing said microencapsulated microorganisms including treating to induce a detectable difference between microcapsules containing mutant microorganisms and those containing non-mutant microorganisms; and (c) separating said microcapsules containing mutant microorganisms from those containing non-mutant microorganisms based on said difference.

FIELD OF THE INVENTION

The present invention relates to a method of isolating mutantmicroorganisms from a population containing the same bymicroencapsulation techniques and to kits for practicing the methods ofthis invention.

BACKGROUND OF THE INVENTION

The enormous size of microbial populations has proved to be a greatasset in a variety of studies, but only because it is possible toeffectively select certain kinds of rare gene type or mutantmicroorganisms. Mutant varieties of a single strain of microorganism(procaryotic, eucaryotic or viral) have classically been isolated by avariety of methods including positive cell "selection" and differential"screening".

Selection is used to isolate mutant varieties of microorganisms when agenetic alteration provides the microorganism with a positive growthadvantage over its parental population. For example, acquisition ofantibiotic resistance can be used to select such mutants on a nutrientagar surface containing the antibiotic. Another example is theacquisition of a biosynthetic gene enabling the organism to grow in aculture medium that would not otherwise support growth. There are,however, other genetic alterations such as additions, substitutions ordeletions of the microorganism's genome which affect the primary orsecondary metabolism of the microorganism, causing a small change ornegative change (decrease) in the rate of growth. Such alterations mayresult in a beneficial increase or decrease in the synthesis or thebreakdown of chosen biochemicals. Under such circumstances, screeningmust generally be utilized to isolate the mutant colony. Screening mayinvolve examination of tens of thousands of individual colonies todetermine the presence of mutants. Replica plating is one such screeningtechnique. In general, it can be said that screening techniques,although highly effective in achieving the desired result, are labor andmaterial-intensive requiring examination of many individual coloniesusually in petri dishes; replica plating and tedious visual comparisonof petri dish pairs are required as well as relatively large amounts ofselective and/or restrictive materials which serve to differentiate themutant from its parent.

Recently, a technology has emerged which provides methods ofencapsulating biological material such as living tissue, individualcells, viruses, and biological macromolecules within a semipermeablemembrane. The basic approach in this technique involves suspending thebiological material to be encapsulated in a physiologically compatiblemedium containing a water soluble substance that can be made insolublein water, that is, a gel, to provide a temporary environment for thebiological material. The medium is formed into droplets containing thetissue and gelled by changing any one of the variety of ambientconditions. These temporary capsules are then subjected to a treatmentwhich results in the production of membranes with a desired permeability(including impermeable membranes). One such technique, is exemplified inU.S. Pat. No. 4,352,883 entitled "Encapsulation of Biological Material",the disclosure of which is incorporated herein by reference.

A description of a technique for separating cells having desiredproperties from a larger population is found in U.S. Pat. No. 4,401,755entitled "Process for Measuring Microbiologically Active Material" whichdiscloses a method for measuring an unknown quantity ofmicrobiologically active material utilizing a microencapsulationtechniques similar to U.S. Pat. No. 4,352,883. The disclosure of U.S.Pat. No. 4,401,755 is also incorporated herein by reference. Afterpreparing a suspension of gel microdroplets, the suspension is processedin an apparatus having the capability of sensing a physicalcharacteristic of individual gel microdroplets to determine the presenceor absence of a desired physical characteristic of the biologicalmaterial in such a droplet.

Microencapsulation technology, as described in the above referencedpatents, provides the potential for solving a variety of problemsincluding the labor and cost excesses of prior art mutant microorganismisolation techniques. It is apparent that a need to develop newisolation techniques exists which will reduce the costs and time spentin selection and screening processes used to isolate mutants from theirrespective parent populations.

SUMMARY OF THE INVENTION

The present invention provides a method of isolating a mutantmicroorganism from it's parent population in the laboratory or in amixed multispecies population such as encountered in agricultural orindustrial environment, or in fermentation. Single or small numbers ofmicroorganisms from the parent population (containing the mutant whichis desired to be isolated) are encapsulated in a semi-permeable membraneby means of microencapsulation techniques. Thereafter, the cells arecultured and the microcapsules containing substantially pure clones ofmicroorganisms are treated to induce a detectable difference (e.g.change in number of microorganisms per microcapsule) betweenmicrocapsules containing the desired mutant microorganisms and thosecontaining non-mutant microorganisms. This detectable difference (suchas provided by a difference in cell number) serves to enable thediscrimination and/or separation of microcapsules containing mutantsfrom those with non-mutants. Finally, the microcapsules containingmutant microorganisms are isolated from those microcapsules containingnon-mutant microorganisms by separation techniques based directly orindirectly on the detectable difference, e.g. a difference inmicrocapsule density or mass resulting from a change in cell number permicrocapsule.

A non-inclusive list of characteristics which can be the basis ofidentifying and/or separating microencapsulated mutant cells from theparental population are increased or decreased cell growth rate, celldensity, cell size, level of synthesis of a detectable primary orsecondary metabolite, level of accumulation of chemical elements orcompounds containing these elements, rate of breakdown of designatedchemicals, antibiotic resistance and various combinations of theseproperties.

For instance, the difference in cell number between microcapsulescontaining mutant and non-mutant cells permits any one of a variety ofseparation techniques. These include both simultaneous (bulk or in toto)separation techniques as well as sequential (or serial) separationtechniques. Examples of bulk separation include equilibrium densitycentrifugation, velocity or gravity sedimentation, separation inelectrical or magnetic fields, chromatography, etc. Examples of serialtechniques include detection of individual microcapsules viaradioactive, luminescent, fluorescent or colorimetric labels, etc.

The present invention overcomes many of the prior art problemsassociated with isolating mutant microorganisms from parent populationsin that, inter alia, it provides in some instances for separation ofmicrocapsules containing the mutants from the microcapsules containingnon-mutants without the necessity for screening techniques which involveindividual examination of microorganisms and therefore can eliminate thelabor and cost excesses of prior art isolation techniques. In otherinstances, by the increasing number of cells in microcapsules containingmutant cells a particular physical characteristic is amplified so thatthe mutant can more easily be separated from a parental population(where the parental population also exhibits that characteristic, but toa lessor degree).

DESCRIPTION OF THE INVENTION

In accordance with the present invention, a method is provided forisolating mutant microorganism from a parent microorganism populationcontaining this same mutant. The method utilizes the relatively newtechnique of microencapsulation of biological material.

In one embodiment, the method of the present invention comprises first,microencapsulating in a semi-permeable membrane individualmicroorganisms in the microorganism population which contains the mutant(desired to be isolated). This enables single mutant microorganisms todivide within a physical envelope so that whatever physical, chemicaland/or biological identity they possess as single mutants (either aconstant identity or an inducible property) can be amplified so as tofacilitate the physical separation and recovery of the desired mutant.

Encapsulation of single microorganisms can be accomplished by techniqueswell known to those skilled in the art and, as will be appreciated,results in microcapsules which are clonally pure. In particular, singlemicroorganism encapsulation can be readily achieved by controlling theconcentration of the microorganism in suspension such that eachmicrocapsule will receive, on average, one microorganism. Generally, themicroorganisms to be encapsulated are prepared in accordance with wellknown prior art techniques, as individual (disaggregated) cells, andsuspended in an aqueous medium suitable for maintaining viability andfor supporting the ongoing metabolic processes of the particularmicroorganism involved. Media suitable for this purpose are availablecommercially. Similarly small numbers of microorganisms can beencapsulated in one microcapsule, if so desired.

The microcapsules are formed so that there is a high probability thateach microcapsule contains a small number or one unit ofmicrobiologically active material (i.e. microorganism or cell). This canbe effected by regulating the dilution of the liquid composition to beused to produce the microcapsules using knowledge of the size of themicrobiologically active material and the predetermined size of themicrocapsule to be produced. The regulation of these factors can bedetermined by conventional Poisson statistical analyses so that thenumber of microcapsules containing more than the desired number ofmicrobiologically active materials is more than two standard deviationsfrom the mean. It is desirable, for example, to encapsulate zero to onemicrobiologically active cell per microcapsule in mutant screening andin recombination DNA research (where the object is generally to isolatedesirable spontaneous mutant microorganisms or genetically engineeredmicroorganisms from a large parental population of such microorganisms).

The preferred encapsulation technique is that described in theabove-referenced U.S. Pat. No. 4,352,883 (Lim). In brief, this approachinvolves suspending the microorganism to be encapsulated in aphysiologically compatible medium containing a water soluble substancethat can be made insoluble in water (gelled) to form a temporaryprotective environment for the microorganisms so encapsulated. Themedium is next formed into droplets containing the single microorganismand gelled, for example, by changing ambient conditions such astemperature, pH or the ionic environment. The "temporary capsules"thereby produced are then subjected to a treatment that results in theproduction of a membrane of a controlled permeability about theshape-retaining temporary capsules.

The temporary capsules can be fabricated from any non-toxic, watersoluble substance that can be gelled to form a shape-retaining mass by achange of conditions in the medium in which it is placed, and that alsocomprises plural groups which are readily ionized to form anionic orcationic groups. The presence of such groups in the polymer enablessurface layers of the capsule to be cross-linked to produce the desiredmembrane when exposed to polymers containing multiple functionalities ofthe opposite charge.

The presently preferred material for forming the temporary capsules is apolysaccharide gum, either natural or synthetic, of the type which canbe (a) gelled to form a shape-retaining mass by being exposed to achange in conditions such as a pH change or by being exposed tomultivalent cations such as Ca⁺⁺ ; and (b) "cross-linked" or hardened bypolymers containing reactive groups such as amine or imine groups whichcan react with acidic polysaccharide constituents. The presentlypreferred gum is alkali metal alginate. Other water soluble gums whichcan be used include guar gum, gum arabic, carrageenan, pectin,tragacanth gum, zanthan gum or acidic fractions thereof. Whenencapsulating thermally refractory materials, gelatin or agar may beused in place of the gums.

The preferred method of formation of the droplets is to force thegum-nutrient-tissue suspension through a vibrating capillary tube placedwithin the center of the vortex created by rapidly stirring a solutioncontaining a multivalent cation. Droplet ejected from the tip of thecapillary immediately contact the solution and gel as spheriodal shapedbodies.

The preferred method of forming the desired semi-permeable membraneabout the temporary capsules is to "cross-link" surface layers of agelled gum of the type having free acid groups with polymers containingacid reactive groups such as amine or imine groups. This is typicallydone in a dilute solution of the selected polymer. Generally, the lowerthe molecular weight of the polymer, the greater the penetration intothe surface of the temporary capsule, and the greater the penetration,the less permeable the resulting membrane. Cross-links are produced as aconsequence of salt formation between the acid reactive groups of thecross-linking polymer and the acid groups of the polysaccharide gum.Within limits, semipermeability can be controlled by selecting themolecular weight of the cross-linking polymer, its concentration, andthe duration of reaction. Cross-linking polymers which have been usedwith success include polyethylenimine and polylysine. Molecular weightcan vary, depending on the degree of permeability required, betweenabout 3,000 and 100,000 or more. Good results are obtained usingpolymers having an average molecular weight on the order of 35,000.

Optionally, with certain materials used to form the temporary capsules,it is possible to improve mass-transfer within the capsule afterformation of the desired membrane by re-establishing the conditionsunder which the material is liquid, e.g., removing the multivalentcation. This can be done by ion exchange, e.g., immersion in phosphatebuffered saline or citrate buffer. In some situations, such as where itis desired to preserve the encapsulated tissue, or where the temporarygelled capsule is permeable, it may be preferable to leave theencapsulated gum in the cross-linked, gelled state.

An alternative method of membrane formation involves an interfacialpolycondensation or polyaddition reaction. This approach involvespreparing a suspension of temporary capsules in an aqueous solution ofthe water soluble reactant which includes a pair of complementarymonomers which can form a polymer. Thereafter, the aqueous phase issuspended in a hydrophobic liquid in which the complementary reactant issoluble. When the second reactant is added to the two-phase system,polymerization takes place at the interface. Permeability can becontrolled by controlling the makeup of the hydrophobic solvent and theconcentration of the reactants. Still another way to form asemi-permeable membrane is to include a quantity of protein in thetemporary capsule which can thereafter be cross-linked in surface layersby exposure to a solution of a cross-linking agent such asgluteraldehyde.

The second step of the method of the present invention preferablycomprises growing the microencapsulated microorganism under conditionswhich induce a difference in the number of microorganisms per capsulebetween microcapsules containing mutant microorganisms and thosecontaining non-mutant microorganisms. The detectable difference can be aresult of the difference in cell number, per se, or the cells can betreated to amplify the difference between mutant clones and parentalclones. That is, within the last three generations of growth or, in somecases, after the microorganisms within the microcapsules have beengrown, they can be further treated in order to amplify particularcharacteristics in the material to facilitate identification andisolation of microcapsules containing mutants. Methods of treatmentinclude incubation, incubation with heavy isotope or radioactive isotopemetabolites, staining with fluorescent stains, labeling withmagnetically tagged substances or immunological agents, etc.

In one embodiment of the present invention the microorganisms are grownunder non-restrictive non-selective conditions within the microcapsulesfor several generations to a predetermined microorganism density toestablish the microorganism's viability and the appropriate populationnumber within each microcapsules. By way of example, singlemicroorganisms are grown, using complete medium, to no more than about5-10% of the final density within the microcapsule. At this point, it ispossible, if desirable, to eliminate empty microcapsules and thosecontaining non-viable or very slow growing organisms by for exampledifferential density-sedimentation, because microorganisms are heavierthan water. If the desired mutant is slow growing, the separation isaccomplished at this point. Thereafter, the microcapsules are grownunder conditions which would induce a difference in mass between mutantand non-mutant containing microcapsules. For example, restrictive and/orselective conditions could be employed during this stage of growth toinduce the desired difference in cell number per capsule. If the mutantdesired to be isolated, for example, is characterized in that it hasacquired antibiotic resistance, then the cells within microcapsules canbe cultured in a medium containing the specific antibiotic. This resultsin mutant growth within microcapsules while further growth of non-mutantstrains in microcapsules would be prevented. If, on the other hand, themutant requires a particular amino acid to grow, restrictive conditionscan be used which will result in mutant-containing microcapsulespossessing fewer microorganisms than non-mutant-containingmicrocapsules, hence providing the desired cell number differentiation.

This has obvious industrial importance. In isolating mutants of yeasts,molds, single cell bacteria, and actinomycetes, in which the mutantsoverproduce valuable enzymes and primary metabolites, it has been notedthat growth of such mutants is usually slower than the parental cells.This is because unbalanced metabolism, if not producing cell toxicity,results in energy waste and nutrient limitations. These mutants, whichare normally hidden in the population, can be selected in accord withthe present invention by, for example, a microcapsule densitycentrifugation protocol. Moreover, the growth rate difference andresulting difference in cell number (which is the basis for theseparation) between microcapsules containing mutant and non-mutantmicroorganisms may be accentuated further by a chosen feeding regimen.Depending on the metabolic process desired to be de-regulated in themutant, an appropriate nutrient source (i.e., carbon, nitrogen,phosphate, or other essential nutrient) can be made limiting. This samegrowth rate selection protocol can be used to functionally differentiatemutations in genetically engineered organisms in which, out of aspectrum of mutations, very few result in enzyme or metaboliteoverproduction.

In another embodiment, mass differentiation between mutant andnon-mutant-containing microcapsules can be enhanced by transferring themicrocapsules to culture medium containing heavy isotope metabolitesincluding, for example, deuterium oxide and/or ¹⁵ N-labeled compoundsfor an appropriate period of time. Subsequently, the microcapsules arewashed in normal culture medium such that the cells within themicrocapsules selectively retain heavy isotopes. This procedure servesto increase the overall density difference between microcapsulescontaining many as compared to those containing few cells.

In still another embodiment, mass differentiation between mutant andnon-mutant-containing microcapsules can be established by inversion ofthe density of the microcapsules. This is accomplished by incubating themicrocapsules in any non-toxic high density medium of appropriatediffusion rate. The microcapsules containing fewer microorganisms willexchange more volume of the medium than microcapsules containing thegreater number of microorganisms, thus becoming more dense andestablishing the desired difference in mass.

Another distinct basis for density separation of encapsulated mutantmicroorganisms from the encapsulated parental microorganism populationis the difference in the intrinsic density of the microorganism itself.If, for example, a mutant microorganism accumulates a heavy metal inelemental or ionic form more efficiently than the non-mutant then, aftergrowth in the presence of such a metal, the microcapsule containing themutants would be more dense. Similarly, other metabolites, periodicelements, or compounds thereof, which accumulate either in themicroorganism or in the microcapsule and change the microcapsuledensity, can provide the basis of separation based on difference in massbetween the various microcapsules.

The final step required in practicing the present invention comprisesseparating the microcapsules containing mutants from those containingnon-mutants based on the detectable difference, such as the differencein number of cells per capsule therebetween. Any one of a variety oftechniques well-known to those skilled in the art may be employed toeffect the desired separation. In particular, where applicable, it ispreferable to use equilibrium density centrifugation, or alternativelyvelocity sedimentation. Electrical or magnetic field separationprotocols, or a combination thereof can also be used to effectseparation. Automated laser cell sorting devices can similarly beutilized to sort microcapsules containing differing numbers of cells. Ifcells are, for example, uniformly labeled with a fluorescent dye thenmicrocapsules containing greater numbers of cells will fluoresce with agreater intensity than microcapsules containing fewer cells. Thus,microcapsules containing mutant cells are separable from microcapsulescontaining non-mutant cells.

The above approach to mutant isolation is revolutionary in severalsenses. Once microcapsule size and porosity are chosen, the isolation ofmutant microorganisms becomes a simple task. In a preferred embodiment,centrifugation replaces visual scanning of a field or any othercollection of microorganism colonies. Isolation of single microcapsulescontaining desirable mutants is accomplished by, for instance,equilibrium density centrifugation. Once the centrifugation parameter isestablished for a species of microorganism, the isolation of almost anymutant of that species is facilitated.

Conditional lethal mutations can be difficult to isolate, however,because the microencapsulation method relies on non-growth, yetsurvival, of such mutants under restrictive growth conditions forrecovery of the mutants. Many conditional lethal mutations may, however,still be isolated by the method of the present invention, because onlyone microorganism out of the many present in each microcapsule at thetime of shift to restrictive growth conditions need survive.

Microorganisms for which the method of the present invention is usefulinclude procaryotic cells--such as, for example, microorganismsincluding single cell bacteria, spores, and actinomycetes, eucaryoticcells--such as yeasts, molds and higher plant and animal cells includingfused cell hybrids such as antibody-producing hybridoma cells, andvirally-infected cells. A non-inclusive list of cell identities whichcan be the basis of identifying mutant cells are increased or decreasedgrowth rate, cell density, cell size, level of synthesis of a detectableprimary or secondary metabolite, level of accumulation of chemicalelements, or compounds of these elements, rate of breakdown ofdesignated chemicals, antibiotic resistance and various combinations ofthese properties.

In another embodiment. a hybridoma cell mixture (which may have beenpreselected for fused (hybridoma) cells with antibiotic or nutrientregimen to eliminate unfused parental cells) is suspended at a cellconcentration such that each microcapsule will receive, on average, onehybridoma cell. For extremely rare monoclonal antibody selection it maybe desirable to originally encapsulate a "pool" of cells, i.e., 2-50hybridoma cells per microcapsule. Following growth and antibodyexpression within the microcapsules (suspended in appropriate nutrientmedium) the microcapsules are screened for specific antibody productionby employing a challenge antigen.

The antigen may be fluorescent or radioistope-labeled such thatfollowing incubation of the microcapsules in the presence ofmicrocapsule-diffusible (small) antigen and thorough rinsing to removeunbound antigen, the specific binding of labeled antigen to antibody canbe easily monitored. In the case of antigens larger than the effectivemicrocapsule pore size and therefore not microcapsule-diffusible, thelabeled antigen is first broken down by mechanical or enzymatic cleavageto a diffusible size and then employed in the screening.

This has enormous potential as a research tool for the followingreasons. For example, if an objective is to generate monoclonalantibodies against surface determinants on a patient's cancer cells, thecancer cells or their outer membranes would be injected into a mouse toelicit antibody response. Later the individual mouse spleen cell-myelomacell hybrids would be microencapsulated and grown to appropriate densitywithin the microcapsules. Hybridoma cell-monoclonal antibody withinthese microcapsules cannot be challenged with whole cancer cells.Rather, the surface components of the intact cancer cells would be madefluorescent or radiolabeled. The cancer cells would then be broken, thelabeled membrane pelleted, and this membrane then broken and/orenzymatically digested to reduce the labeled surface components tomicrocapsule-diffusible size. These labeled components would then beutilized for microcapsule monoclonal antibody screening. Themicrocapsules containing different monoclonal antibodies optionally canbe pre-incubated (pre-competed) with the unlabeled (non-fluorescent andnon-radioactive) cell surface components of the respective non-cancerouscells. Such pre-competing reduces the "false-positive" microcapsules,i.e. those not producing cancer-specific antibodies.

One can also differentially label (by isotope and fluorescentderivatives) different classes of macromolecules on the cancer cellsurface such as protein, carbohydrate or lipid. One can then distinguishmicrocapsules containing antibodies directed against the differentclasses of cellular macromolecules.

Following the binding of labeled antigen to antibody within themicrocapsule, residual unbound antigen is washed from the microcapsulesusing appropriate buffer or culture medium. The desired microcapsulescontaining radioactive and/or fluorescent bound antigen are physicallyseparated from the gross population of microcapsules. Most easilyseparated are fluorescent spheres which can be harvested using anautomated laser cell sorter typically employed to separate T and Blymphocyte cells. Rapid screening of radioactive (as well asfluorescent) microspheres can also be accomplished by a very differentand less expensive method. First, an 8×10 inch "monolayer" of spherescan be deposited on a plate (each sphere covers approximately 0.01 mm²).This layer will contain approximately 5×10⁶ microspheres. Photographicemulsion-autoradiography can be utilized to locate the radioactivemicrospheres. Since these microspheres are on the order of 100 microns(0.1 mM) in diameter their autoradiographic image should be visible onfilm. A luminescent reference grid included in the microsphere supportsurface helps the investigator to align the film with the microspheresupport surface and identify a small region containing the radioactivemicrosphere. Suction aspiration of this region yields a small number ofmicrospheres. These small groups of microspheres are spread out andagain autoradiographed to yield individual desired microspheres. Forfluorescent antigen applications, the microspheres are briefly exposedduring photography to UV light. Again a luminescent or fluorescentspacial reference grid is included to align the film with themicrosphere support surface. The support surface between microspheresand film is preferably sufficiently thin to minimize angular dispersalof microsphere source radiation.

Current methods for selecting new and commercially important strains ofmicroorganisms for industrial process usually involve screening singlecells for altered physical or biochemical properties (under themicroscope) or screening multicellular colonies derived from singlecells for new biochemical properties, or for increases or decreases inexisting biochemical abilities. This is particularly difficult to dowhen the microorganism is a member of a complex culture or ecosystemconsisting of more than one species of live organism (said complexculture being also termed "non-sterile"). Furthermore, the property orproperties of the desired strain (i.e. "mutant") may depend uponconcerted growth of more than one microorganism in the non-sterilemedium. Some examples of such non-sterile culture environments includeactivated-sludge process for sewage treatment, fruit and grain mashprocess, biomass fermentation, dairy process, petrochemical process,mineral leaching process, soil microorganism growth (nitrogen fixation,etc.), swamp and lake eutrification process, etc. In accord with themethod of the present invention in order to obtain a mutant of aparticular microorganism strain for industrial process, themicroorganism is microencapsulated as described above. After incubatingthe microcapsules in the complex culture environment, the microcapsulesare recovered and those containing the desired mutant cells are selectedas described herein. Valuable new mutants of desired species can thus beisolated from such complex environments. One can select, screen, andrecover mutant cells which have grown to microcolonies within themicrocapsules while they are in "communication" via diffusion with thecomplex environment.

It is easy to see that state of the art isolation of plant or animalcell mutants, e.g. mammalian cells in tissue culture, is revolutionizedby the method of present invention. This envisions encapsulation ofsingle cells, growth of all cells for a few generations withoutselection, and finally completion of growth under the restrictive and/orselective conditions. This procedure is preferably followed by thedensity-centrifugation separation of mutant from wild-type cells.Nutritional cross-feeding of procaryotic or eucaryotic mutants bywild-type cells is not a problem. To the contrary, the mutant andwild-type clones are physically separated from each other by themicrocapsule membrane as are colonies on agar. More importantly, becausethe selective phase typically lasts about five to seven generationsusing microcapsules, any microcapsule colony cross-feeding problems arereduced compared with conventional colonies experiencing longerincubations.

In still yet another embodiment, other substances may be included alongwith the microorganism during the microencapsulation step. For instance,it may be desirable to include growth hormones such as fibroblast growthfactor and/or epidermal growth factor, other non-diffusible growthfactors (typically proteins or glycoproteins) or adhesion surfaces, suchas fibrinogen, collagen, etc.

For example, adhesion surfaces (or substratum), such as microcarrierbeads, may be encapsulated together with the microorganisms. Certaincells require an adhesion surface for growth. The encapsulation ofmicrocarrier beads as the substratum, each bead carrying a single cell,prevents cross-contamination of microorganisms which would otherwiseoccur between non-encapsulated carrier beads. The encapsulation ofmicroorganisms on beads is particularly useful in growing microorganismssuch as normal and malignant mammalian cells which often require thepresence of solid substratums. For example, in screening potentialanti-cancer drugs, such microencapsulation allows one to follow theinhibition of growth and development of individual cancer cells in thepresence of chosen drug regimens.

The present invention also provides the means for solving other priorart problems. For example, conventional agar methods of selectingmutations conferring positive growth advantage have been previouslymentioned. When, however, the parental cells exhibit "leaky" growth orgrow at a rate just 10-20% slower than the desired mutant, the selectionmay become problematic. In such a case repeated serial "passage" of aculture may be attempted to enrich for the faster growing mutant.However, if positive selection is performed in microcapsules and inaccord with the present invention, the following would be expected. A200 um diameter microcapsule (4×10⁻⁶ ml volume) is formed containing onemutant cell. This cell is a bacterium which in normal medium grows to acell density of 10¹⁰ cells/ml or about 4×10⁴ cells/microcapsule. Thisrepresents 15 cell doubling (generations). If the mutant's growth isonly 10% faster than the parent, then after 14 generations, the "mutant"microcapsules will contain twice as many cells as the othermicrocapsules. This can provide a sufficient basis for physicalseparation. The same mutant cells grown by serial passage selection forthe same number of generations would simply be enriched two-fold withinthe bulk parental population. Using calculations, one can rapidlyestimate the ability to isolate mutants of other microorganisms given amodest growth rate differential. Thus, the advantages of the presentinvention are readily apparent.

The present invention also provides a kit for practicing the methodsdescribed above. The kit comprises the ingredients or componentsrequired to form microcapsules on a laboratory or research scale toscreen for mutants in accord with the present invention. As such, thekit comprises a container or package having quality-controlled reagentstherein: sterile alginate solution, sterile 2-(cyclohexylamino)ethanesulfonic acid, sterile solution of polylysine having a predeterminedmolecular weight and a sterile solution of polyethylenimine (PEI) havinga predetermined molecular weight. The molecular weights of thepolylysine and PEI are predetermined to produce microcapsules having adesired permeability in accord with the known technology as describedin, for instance, U.S. Pat. No. 4,352,883. In addition, sterile CaCl₂and mechanical devices for forming microdroplets can be supplied as partof the kit. Preferably, the solutions are provided in sealed sterilevials having sufficient quantities for one experiment. Further, it ispreferred that the solutions comprise physiological saline and thepolylysine and PEI solutions also contain 0.2M MOPS[3-(N-morpholino)propanesulfonic acid] buffer (pH6).

From the foregoing it will be apparent that isolation of mutantmicroorganisms in accordance with the present invention can be practicedon a wide variety of organisms, using: (1) a wide variety of techniquesto induce the difference in number of microorganisms per capsule betweenmutant and non mutant-containing microcapsules, and (2) a wide varietyof separation techniques to isolate the desired mutant without departingfrom the scope and spirit of the invention.

It is appreciated that those skilled in the art, upon consideration ofthis disclosure, may make modifications and improvements within thespirit and scope of this invention.

What is claimed is:
 1. A method for isolating a mutant microorganism, which method comprises the steps of:a. separately microencapsulating in a semi-permeable membrane each or a small number of microorganisms from a microorganism population containing said mutant; b. growing said microencapsulated microorganisms and treating them to induce a difference in cell number between microcapsules containing mutant microorganisms and those containing non-mutant microorganisms; and c. separating said microcapsules containing mutant micoorganisms from those containing non-mutant microorganisms by a method based on said difference in cell numbers.
 2. The method of claim 1, wherein said mutant microorganism is a naturally occuring mutant of a wild type microorganism.
 3. The method of claim 1, wherein the mutation in the mutant microorgahism is artificially induced.
 4. The method of claim 3, wherein said artificially-induced mutant is the result of genetic engineering by a biological, biochemical or biophysical process.
 5. The method of claim 1, wherein said microcapsules containing said single microorganisms are formed by:a. forming a dilute suspension of said microorganisms in a liquid diluent capable of forming a gel upon said dilute suspension having a dilution selected so that there is a high probability that each microcapsule produced from said suspension contains one microorganism; b. converting said suspension into gel droplets, forming said microcapsules.
 6. The method of claim 1, wherein said treatment comprises growing said microencapsulated microorganisms under conditions restrictive to said mutant whereby said microcapsules containing said mutants have fewer microorganisms per microcapsule than microcapsules containing non-mutants.
 7. In the method of claim 6, prior to said separation step, incubating said microcapsules in a high-density medium of appropriate diffusion rate, thereby causing the mutants in the microcapsules which have fewer microorganisms to have a higher density than the non-mutants in the microcapsules which have larger numbers of microorganisms.
 8. The method of claim 1 wherein in said growing step (b) the microencasulated mutants have a capacity to accumulate a particular material resulting in an increase in mass and number greater than that of said microencapsulated non-mutants.
 9. The method of claim 1, wherein said separation is based on the difference in mass between said microcapsules containing mutant and non-mutant microorganisms and comprises (a) equilibrium density centrifugation of a suspension containing said microcapsules or (b) velocity sedimentation of a suspension containing said microcapsules.
 10. The method of claim 1, wherein said microorganisms comprise eucaryotic cells.
 11. The method of claim 1, wherein said microorganisms comprise procaryotic cells.
 12. The method of claim 1, wherein said microorganisms comprise viruses.
 13. The method of claim 1, wherein said microorganisms comprise hybridoma cells.
 14. The method of claim 1, wherein mutant microorganisms are selected from chemically complex and non-sterile agricultural, industrial or other commercial process environments.
 15. The method of claim 1, wherein said microencapsulated microorganisms are grown in a laboratory culturing medium.
 16. The method of claim 1, wherein said microencapsulated microorganisms are grown in an agricultural or industrial process medium wherein one or more species of said microorganism are found in the native environment. 